Many hormones and neurotransmitters mediate biological functions through specific receptors present on the cell membrane. Many of these receptors engage themselves in the intracellular transduction of signals through activation of the coupled guanine nucleotide-binding protein (hereinafter sometimes referred to briefly as G protein) and have the common structure comprising 7 transmembrane domains. Therefore, these receptors are collectively referred to as G protein-coupled receptor or 7-transmembrane receptor.
Through the interaction of such hormones or neurotransmitters with G protein-coupled receptors, a variety of regulatory functions of vital importance, for example maintenance of homeostasis, reproduction, development, metabolism and growth, and regulation of the nervous system, circulatory system, immune system, gastrointestinal system and metabolic system are all discharged. While it is known, as mentioned above, that receptor proteins exist for various hormones or neurotransmitters and are playing important roles in the control of vital functions, it is still unknown in many respects whether unknown active substances (hormones or neurotransmitters) and their receptors ever exist or not.
In recent years, by taking advantage of the similarity of such G protein-coupled receptor proteins in partial amino acid sequence, searches for DNAs coding for novel receptor proteins have been undertaken by the polymerase chain reaction technique (abbreviated as PCR) and actually a large number of orphan G protein-coupled receptors, so called because their ligands are unknown, have been cloned to this day (Libert, F., et al., Science, 244, 569-572, 1989; Welch, S. K., et al., Biochem. Biophys. Res. Commun., 209, 606-613, 1995; Marchese, A., et al., Genomics, 23, 609-618, 1994; Marchese, A., Genomics, 29, 335-344, 1995). Furthermore, as the result of random sequencing of genomic DNAs and cDNAs, novel G protein-coupled receptor proteins have been discovered one after another (Nomura, N., et al., DNA Research, vol. 1, 27-35, 1994). The only general means so far available for determining the ligands to such orphan G protein-coupled receptor proteins is a mere estimation based on the similarity in primary structure among G protein-coupled receptor proteins. However, many orphan G protein-coupled receptor proteins have low homology with known receptors and it is, therefore, difficult to estimate their ligands based on the similarity in primary structure alone except in those cases where they are actually subtypes of receptors for known ligands. On the other hand, since many orphan G protein-coupled receptors have been discovered as the result of gene analysis, it is supposed that there still exist many unknown corresponding ligands. However, only in a few cases, ligands have actually been identified for such orphan G protein-coupled receptors.
Recently, investigation for novel opioid peptides by introducing a cDNA coding for a receptor protein which a ligand is unknown, i.e. an orphan G protein-coupled receptor protein, into animal cells have been reported (Reinsheid, R. K. et al., Science, 270, 792-794, 1995, Menular, J.-C., et al., Nature 377, 532-535, 1995). However, in view of similarities to known G protein-coupled receptor proteins and tissue distributions, it could be easily anticipated in these cases that the ligand would be belonging to the family of opioid peptides. The history of research and development in the realm of substances acting on the living body through the opioid receptor dates back to many years ago and various antagonists and agonists have been developed. Therefore, among the compounds artificially synthesized, an agonist of the receptor was picked out and, using it as a probe, expression of the receptor in the receptor cDNA-transfected cells was verified. Then, a search was made for an activator of the intracellular signal transduction which was similar to the agonist, the activator so found was purified, and the structure of the ligand was determined. However, when the homology of an orphan receptor to known G protein-coupled receptor proteins is low, it was very difficult to predict its ligand.
APJ is one of the orphan G protein-coupled receptors so far reported (O'Dowd, B. F., et al., Gene, 436, 355-359, 1993). APJ has low homology with an angiotensin receptor (AT1) but its ligand has remained unknown since no reaction was detected at all with angiotensin II despite the expression of APJ in CHO cells.
Ligands for APJ, which is an orphan G protein-coupled receptor expressed in the central nervous system, circulatory system, reproductive system, immune system, digestive organs and so forth, are expected to be useful as drugs but their structure and functions have not been elucidated as yet.